Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Further-more, the A-chain features one intra-chain disulphide bridge.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains to form the two-chain insulin molecule. Insulin is essential in maintaining normal metabolic regulation.
The two chain structure of insulin allows insulin to undertake multiple conformations, and several findings have indicated that insulin has the propensity to considerable conformational change and that restrictions in the potential for such change considerably decrease the affinity of the insulin receptor for ligands. Proinsulin has a 100 fold lower affinity for the insulin receptor than native insulin. Blocking of the amino acid residue A1 in insulin also results in poor receptor binding, consistent with the dogma that a free N-terminal of the A-chain and free C-terminal of the B-chain of insulin are important for binding to the insulin receptor.
The inherited physical and chemical stability of the insulin molecule is a basic condition for insulin therapy of diabetes mellitus. These basic properties are fundamental for insulin formulation and for applicable insulin administration methods, as well as for shelf-life and storage conditions of pharmaceutical preparations. Use of solutions in administration of insulin exposes the molecule to a combination of factors, e.g. elevated temperature, variable air-liquid-solid interphases as well as shear forces, which may result in irreversible conformation changes e.g. fibrillation. This is particularly relevant for insulin solutions in infusion pumps, either worn externally or implanted, which exposes the molecule to a combination of these factors as well as shear forces from the movement of the pump for extended periods of time. Consequently, fibrillation is especially a concern when using infusion pumps as insulin delivery system. Moreover, the solubility of insulin is influenced by multiple factors and shows clear reduction in the pH range from 4.2 and 6.6. The pH precipitation zone generally imposes limitations for formulation, but has also been used deliberately in development and formulation of certain analogues.
Thus, the stability and solubility properties of insulin are important underlying aspects for current insulin therapy. The present invention is addressed to these issues by providing stable, single-chain insulin analogues by introduction of a C-peptide between the B- and A-chain to decrease molecular flexibility and concomitantly reduce the fibrillation propensity and limit or modify the pH precipitation zone.
Single-chain insulins with insulin activity are disclosed in EP 1,193,272. These single-chain insulins have a modified C-peptide of 5-18 amino acids and are reported to have up to 42% insulin activity. EP 1,193,272 discloses the following modified C-peptides connecting B30 with A21: Gly-Gly-Gly-Pro-Gly-Lys-Arg(SEQ ID NO:1), Arg-Arg-Gly-Pro-Gly-Gly-Gly(SEQ ID NO:2), Gly-Gly-Gly-Gly-Gly-Lys-Arg(SEQ ID NO:3), Arg-Arg-Gly-Gly-Gly-Gly-Gly(SEQ ID NO:4), Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg(SEQ ID NO:5), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO:6), Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg(SEQ ID NO:7), Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO:8), Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg(SEQ ID NO:9), and Arg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO:10). EP 741,188 discloses single-chain insulins with a modified C-peptide having from 10-14 amino acids residues and having from 14 to 34% insulin activity and having the following connecting peptides Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg(SEQ ID NO:11) and Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr(SEQ ID NO:12). WO 95/16708 discloses single-chain insulins with a connecting peptide of 1-15 amino acid residues and with no Lys or Arg as the C-terminal amino acid residue in the connecting peptide. WO 95/16708 discloses the following C-peptide sequences Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr(SEQ ID NO:13) and Gly-Tyr-Gly-Ser-Ser-Ser-Ala-Ala-Ala-Pro-Gln-Thr(SEQ ID NO:14). These single-chain insulins are reported to have insulin activity but also a fairly high affinity to the IGF-1 receptor.
It is the object of the present invention to provide single-chain insulins which have improved properties over the known compounds both with respect to insulin activity, physical stability and solubility as well as pharmacokinetic e.g. a protracted or rapid action profile. A still further object of this invention is to provide a method for making the single-chain insulins and pharmaceutical compositions containing such compounds.